An essential element in the design of a rational and effective therapy for prion diseases as well as other diseases is the detailed and accurate knowledge of their pathogenesis. The inherited forms of prion diseases offer an easier approach to the study of the pathogenesis and have provided crucial insights applicable to the pathogenesis of the sporadic form which is more prevalent, but more difficult to investigate. The pathogenesis of inherited prion diseases may be viewed as a three stage process. The first includes any potential direct pathogenic effect inherent to the mutant prion protein (PrP). The second are the conversion mechanisms of mutant PrP into protease-resistant PrP. The third are the pathogenic mechanisms of the protease-resistant PrP. This research project addresses all three stages in four Specific Aims. Specific Aim 1 deals with a study of the metabolism of D178N, 129N and 144bp insertion mutant PrP in fibroblasts from patients with fatal familial insomnia and variable phenotype, respectively. Data obtained from patient~s fibroblasts will be compared with findings generated by human neuronal cells from the central nervous system. The purpose of this set of studies is to identify the abnormality of the mutant PrP in these two human prion diseases and in other prion diseases for which fibroblasts are available. Specific Aim 2 focuses on the conversion mechanisms of mutant PrP intoprotese-resistant PrP. This process will be studies in neuroblastoma cells transfected with PrP gene constructs carrying the mutations of human inherited prion diseases. These transfected neuroblastoma cells will be infected by exposure to exogenous protease-resistant PrP isolated from brains of subjects with sporadic and inherited prion diseases. Specific Aim 3 addresses the issue of transfer of protease-resistant PrP among different cell types present in the nervous system such as neuronal, astroglial and microglial cells. Finally, the mechanisms of propagation of protease-resistant PrP within separate brain regions will be evaluated in Specific Aim 4. Transgenic mice overexpressing PrP will be employed to examine the mode of the protease-resistant PrP propagation from the eye to the superior colliculus along the primary optic pathway. Metabolic labeling, immunohistochemistry and autoradiography will be utilized to resolve whether the protease-resistant PrP migrate intraaxonally or along the extracellular space. The proposed studies will generate important information regarding the cellular mechanisms involved in the pathogenesis of prion diseases, a vital step in the formulation of a rational and effective treatment.